Plasmids construction
All enzymes used for cloning procedures were purchased from Takara except T4 DNA ligase
from New England Biolabs and KOD-plus-Neo DNA polymerase from TOYOBO. UL31 ORF
(composed of 813 bp) was amplified via PCR from the genomic DNA of the PRV Becker strain
using a genome previously purified from vBecker2-infected PK-15 cells [3, 5], as the template.
After the PCR amplified product was validated as the intended product, it was purified using a
PCR gel purification kit (Qiagen) according to the manufacturer’s instructions. The product was
then digested with EcoRI and BamHI and inserted into the correspondingly digested green
fluorescent protein variant mammalian expression vector pEYFP-N1 (Clontech) encoding EYFP
at 16 oC overnight using T4 DNA ligase, to create the recombinant plasmid named pUL31-EYFP,
as described previously [2-4] . After mini-scale isolation of plasmid DNA using Qiagen plasmid
Mini kits (Qiagen), the presence of the appropriate insert in the obtained plasmid was confirmed
by PCR, restriction analysis and sequencing. The series of mutant deletions including amino acids
substitution were generated by PCR-ligation-PCR mutagenesis [1] using appropriate primers
(sequences available upon request) and cloned into pEYFP-N1 as described above, all the N
terminal deleted fusion proteins were inserted with an artificial ATG start codon starting at internal
positions in UL31 of the fusion constructs. Finally, all constructs were verified by PCR, restriction
analysis and sequencing.
Transfection and fluorescence microscopy
To express the proteins in vitro, COS-7 cells were plated onto six-well plates (Corning) in
DMEM with 10% FBS at a density of ~2.5×105 cells per well overnight to 60-80% confluency
before transfection. The next day, monolayer of COS-7 cells were transfected with 1.0 to 1.5 ug
plasmid DNA mixed with Turbofet transfection reagent (Thermo Scientific) according to the
manufacturer’s instructions. The transfection efficiency (~60%) was determined by transfection
with EYFP. At 24 h post transfection, cells were washed with fresh growth medium and processed
for fluorescence microscopy. In the same experiment, each transfection was performed for at least
three times. Data shown were from one representative experiment. Samples were analyzed using a
Zeiss Axiovert 200M microscope (Germany). All the photomicrographs were taken under a
magnification of 400×. Each photomicrograph represents a vast majority of the cells with similar
subcellular localization. Fluorescent images of EYFP fusion proteins were presented in
pseudocolor green, and images were processed using Adobe Photoshop.
References
1.
Ali SA, Steinkasserer A (1995) PCR-ligation-PCR mutagenesis: a protocol for creating gene
fusions and mutations. Biotechniques 18:746-750
2.
Li ML, Wang L, Ren XM, Zheng CF (2011) Host cell targets of tegument protein VP22 of
herpes simplex virus 1. Arch Virol 156:1079-1084
3.
Li ML, Wang S, Cai MS, Zheng CF (2011) Identification of nuclear and nucleolar localization
signals of pseudorabies virus (PRV) early protein UL54 reveals that its nuclear targeting is
required for efficient production of PRV. J Virol 85:10239-10251
4.
Li ML, Li Z, Li WT, Wang BY, Ma CQ, Chen JH, Cai MS (2012) Preparation and
characterization of an antiserum against truncated UL54 protein of pseudorabies virus. Acta
Virol 56:315-322
5.
Smith GA, Enquist LW (2000) A self-recombining bacterial artificial chromosome and its
application for analysis of herpesvirus pathogenesis. Proc Natl Acad Sci U S A 97:4873-4878